Anna M. Grabowska

Direct ex vivo comparison of the breadth and specificity of the T cells in the liver and peripheral blood of patients with chronic HCV infection

The role of intrahepatic lymphocytes in the control of hepatitis C virus (HCV) infection and the pathology associated with it is not understood; most studies of the immunology of this infection use peripheral blood lymphocyte populations. To address this further, we examined in detail the IHL from HCV‐infected patients and controls, focusing on the antigen‐specific CD8+ T lymphocyte component. Individual T cells from needle liver biopsies and peripheral blood were isolated from patients with chronic HCV infection and examined directly ex vivo. We used RT‐PCR spectratyping to compare the breadth of the T cell receptor usage in the liver in comparison with the peripheral blood, and applied MHC class I tetramer technology to investigate the numbers of HCV‐specific CD8+ cells in the two compartments. T cell receptor usage in the liver of HCV‐infected patients was broad, comparable with that in the peripheral blood of the same patients. A much higher proportion of liver CD8+ cells expressed receptors specific for HCV antigens compared with paired peripheral blood CD8+ cells. A greater proportion of the liver tetramer‐positive cells expressed the activation marker CD69, compared with those in the periphery or other CD8+ cells in the liver. In the course of chronic HCV infection, HCV‐specific CD8 cells, which have been recently activated, appear to accumulate specifically in the livers of infected patients but are present in much lower numbers in the peripheral circulation. Further studies are needed to determine the function of these cells and their role in protection and immunopathology.

Gastrin — active participant or bystander in gastric carcinogenesis?

Gastrin is a pro-proliferative, anti-apoptotic hormone with a central role in acid secretion in the gastric mucosa and a long-standing association with malignant progression in transgenic mouse models. However, its exact role in human gastric malignancy requires further validation. Gastrin expression is tightly regulated by two closely associated hormones, somatostatin and gastrin-releasing peptide, and aspects of their interaction may be deregulated during progression to gastric adenocarcinoma. Furthermore, agonists and antagonists of the receptors for all three hormones have shown modest clinical efficacy against gastric adenocarcinoma, which might provide useful information on the future combined use of these agents.

Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA

The recent development of tagged RT-PCR and rTth RT-PCR has greatly improved strand-specific detection of hepatitis C virus (HCV) RNA but these assays are still prone to some false detection of the incorrect strand of RNA. In this study we aimed to address additional factors which contribute towards false detection of HCV RNA. Firstly the benefits of both tagged primers and the thermostable reverse transcriptase rTth during cDNA synthesis were combined and it was found that strand specificity was greatly improved without compromising sensitivity. The reliability of the assay was then optimised by addressing the following issues: control synthetic transcripts should be free of contaminating plasmid DNA, residual RT activity should be minimised in the presence of PCR primers and cDNA should be free of unincorporated tagged RT primer prior to PCR amplification. The alterations made to the assay eliminated completely false detection of the incorrect strand of RNA in the control assay whilst the correct strand was consistently detected at a cDNA dilution of 10(-3)-10(-4). Negative strand was not detected in RNA isolated from serum but was detected, at a ten-fold lower level than positive strand, in RNA isolated from liver tissue.

TT virus sequence heterogeneity in vivo: evidence for co-infection with multiple genetic types

TT virus (TTV) is a newly described DNA virus of humans that exhibits an unusually high degree of genetic heterogeneity. We have performed extensive analysis of the TTV populations present in samples, taken over a period of 2 to 6 years, from three individuals with persistent TTV infection. TTV DNA titres estimated for sequential samples were found to be quite stable over the entire study period in two patients, but fluctuated considerably in the third. DNA sequence analysis revealed different genetic diversity among TTV populations from samples from the three patients. In one case, absolute sequence homogeneity was observed among samples over a 3 year period. In a second, a limited amount of heterogeneity was found, including one sequence exhibiting G-->A hypermutation. TTV DNA sequences from the third patient exhibited quite remarkable genetic heterogeneity: evidence was found of seven distinct infecting viruses, representing four of the six TTV genotypes that have been described. In addition, minor variants of three of these seven sequences were observed. The heterogeneity of the viral population in this individual declined steadily over a 6 year period. This patient infected with a genetically diverse TTV population had the highest viral DNA titre.

Mutational signatures in esophageal adenocarcinoma define etiologically distinct subgroups with therapeutic relevance

Esophageal adenocarcinoma (EAC) has a poor outcome, and targeted therapy trials have thus far been disappointing owing to a lack of robust stratification methods. Whole-genome sequencing (WGS) analysis of 129 cases demonstrated that this is a heterogeneous cancer dominated by copy number alterations with frequent large-scale rearrangements. Co-amplification of receptor tyrosine kinases (RTKs) and/or downstream mitogenic activation is almost ubiquitous; thus tailored combination RTK inhibitor (RTKi) therapy might be required, as we demonstrate in vitro. However, mutational signatures showed three distinct molecular subtypes with potential therapeutic relevance, which we verified in an independent cohort (n = 87): (i) enrichment for BRCA signature with prevalent defects in the homologous recombination pathway; (ii) dominant T>G mutational pattern associated with a high mutational load and neoantigen burden; and (iii) C>A/T mutational pattern with evidence of an aging imprint. These subtypes could be ascertained using a clinically applicable sequencing strategy (low coverage) as a basis for therapy selection.

Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

Background and aims Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines. Methods Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT. Results The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins. Conclusion The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.

TT Virus Infection in Patients with Hepatitis C: Frequency, Persistence, and Sequence Heterogeneity

TT virus (TTV) was recently identified in the serum of a patient with hepatitis. The role of TTV in liver disease has not been established. Three polymerase chain reaction (PCR) protocols were used to detect TTV DNA in sera of persons infected with hepatitis C virus (HCV) and in blood donors. Sera from 11.5% of HCV-infected patients and 7.7% of blood donors were positive by protocols 1 or 2. In contrast, 48.7% and 57.7% of sera, respectively, were positive when tested by protocol 3. There was no difference in the severity of hepatitis in persons coinfected with TTV and HCV when compared with those infected with HCV alone, regardless of which TTV PCR protocol was used. TTV DNA persisted in serum samples taken up to 6 years apart in individual patients. Sequence analysis indicated that most viral sequences were distinct between patients, and there was evidence of genetic heterogeneity and viral evolution within individuals.

Evolutionary trends of the first hypervariable region of the hepatitis C virus E2 protein in individuals with differing liver disease severity.

Hepatitis C virus (HCV) exists as a complex swarm of genetically related viruses known as a quasispecies. Recent work has shown that quasispecies complexity and evolutionary rates are associated with the outcome of acute infection. Knowledge of how the virus population evolves at different stages of chronic infection is less clear. We have studied rates of evolution of the first hypervariable region (HVR1) of the E2 envelope protein in six individuals with disparate liver disease severity. These data show that virus populations present in individuals with mild non-progressive liver disease evolve in a typical Darwinian fashion, with a consistent accumulation of non-synonymous (amino acid-changing) substitutions. By contrast, the virus population remains relatively static in individuals with severe progressive liver disease. Possible mechanisms for this disparity are discussed.

The Biological and Therapeutic Importance of Gastrin Gene Expression in Pancreatic Adenocarcinomas

The gastrin gene is expressed widely in pancreatic adenocarcinomas and the study aimed to assess its role in both the resistance of cancer cells to apoptosis and the sensitivity of cells to chemotherapeutic agents. Two human pancreatic cell lines, PAN1 and BXPC3, expressed gastrin at both the RNA and protein levels and are shown to be representative of human pancreatic adenocarcinomas in terms of gastrin expression. Inhibition of endogenous gastrin production by tumor cells was achieved with neutralizing gastrin antiserum and transfection with a gastrin antisense plasmid. Gastrin antiserum synergized with both taxotere and gemcitabine in inhibiting the in vitro growth of the PAN1 cell line with the inhibitory effect of the antiserum increasing from 12.7% to 70.2% with taxotere (P < 0.05) and 28.6% with gemcitabine (P < 0.01) after controlling for the effects of the cytotoxics. Synergy was only achieved with taxotere in BXPC3 cells with the inhibitory effect of gastrin antiserum increasing from 22.9% to 50.0% (P < 0.005). Cells transfected with gastrin antisense had reduced in vitro growth in low serum conditions and were poorly tumorigenic in nude mice at an orthotopic site. Gastrin antisense-transfected PAN1 cells had increased sensitivity to the antiproliferative effects of both gemcitabine (IC50 of >100 μg/ml reduced to 0.1 μg/ml) and taxotere (IC50 of 20 μg/ml reduced to <0.01 μg/ml) when compared with vector controls. The increased sensitivity of PAN1 antisense coincided with increased caspase-3 activity and reduced protein kinase B/Akt phosphorylation in response to both gemcitabine and taxotere. Gastrin gene circumvention may be an optimal adjunct to chemotherapeutic agents, such as taxotere and gemcitabine, in pancreatic adenocarcinoma.

Gastrin enhances the angiogenic potential of endothelial cells via modulation of heparin-binding epidermal-like growth factor

This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycine-extended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC(Min) mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin-2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC(Min) mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK-2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy.